title: Polymerase reaction without primers throughout for the reconstruction of full-length cDNA from products of rapid amplification of cDNA ends (RACE).
abstract: Rapid amplification of cDNA ends (RACE) has widely been used to determine both ends of the cDNA from its partial sequence. Conventionally, 5'- and 3'-RACE products were ligated at a restriction site in the overlap region to reconstruct the full-length cDNA; however, reconstruction is difficult if no appropriate restriction enzymes are available. Here, we report a novel method to reconstruct full-length cDNA with DNA polymerase. Instead of usual PCR, chain reactions were avoided and the elongation time was shortened, which enables non-specific products or undesired point mutations to be minimized. We successfully reconstructed and TA-cloned a full-length cDNA of echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene variant 2 from RACE products obtained from a surgically resected lung adenocarcinoma sample. We also evaluated some parameters to provide recommendations for this new method.
authors.affiliation: Department of Respiratory Medicine, University of Tokyo Hospital, Tokyo, Japan.
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Sunohara
authors.forename: Masanori
authors.initials: M
authors.lastname: Kawakami
authors.forename: Hidenori
authors.initials: H
authors.lastname: Kage
authors.forename: Kousuke
authors.initials: K
authors.lastname: Watanabe
authors.forename: Noriko
authors.initials: N
authors.lastname: Emoto
authors.forename: Takahide
authors.initials: T
authors.lastname: Nagase
authors.forename: Nobuya
authors.initials: N
authors.lastname: Ohishi
authors.forename: Daiya
authors.initials: D
authors.lastname: Takai
mesh_terms: D011088:DNA Ligases / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D008967:Molecular Biology / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D004259:DNA-Directed DNA Polymerase; D011088:DNA Ligases